A High Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury during the COVID-19 pandemic

Abstract

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo , Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro , SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.

Figure 1.. High relative expression of MUC1 in human lung.

A. Immunoperoxidase staining of three human lung samples demonstrates MUC1 protein abundance in lung tissue. B. mRNA expression data from three datasets (HPA, GTEx, and FANTOM5, heatmaps) combined into a CONSENSUS normalized transcript expression level (bar plot) show enhanced expression of MUC1 mRNA in human lung. NX, normalized expression; pTPM, protein-coding transcripts per million.

Figure 2.. High Content Screening reveals significant and dose-dependent reduction in MUC1 by the FDA-approved SYK inhibitor R406

A. Screening pipeline. B. Primary screen revealed four major groups of compounds which affected MUC1 levels. MUC1 immunofluorescence (IF) signal intensity per cell (normalized to positive control JQ1 minus DMSO-treated controls) plotted vs. DMSO-normalized cell number. Horizontal and vertical dashed lines delineate mean DMSO values +/− 3*SD for both MUC1 intensity and cell number. Lavender-shaded area demarcates candidate MUC1 suppressors. C. qPCR and cell viability profiling screens identified four compounds that reduced MUC1 protein abundance without changing MUC1 mRNA level, and in the absence of cytotoxicity. Left: MUC1 signal intensity per cell plotted vs. MUC1 mRNA level (qPCR assay). Both parameters are normalized to JQ1 minus DMSO-treated controls. Right: JQ1 minus DMSO-normalized MUC1 signal intensity per cell plotted vs. DMSO-normalized cell viability (a number of viable cells after 6 days exposure to the test compounds). Horizontal dashed lines delineate SD for DMSO treated control wells for both cell viability and MUC1 qPCR. Green-shaded areas demarcate candidate hits. D. R406 concentration response curves for MUC1 protein abundance (black), MUC1 mRNA abundance (light gray), and cell viability (dark gray). E. MUC1 IF in kidney epithelial cells treated for 48 hours in the absence (DMSO) and presence of R406 at the indicated concentrations.

Figure 3.. R406 preferentially depletes MUC1 from the plasma membrane.

A. R406 (at EC50 concentration) substantially reduced MUC1 abundance in or near the plasma membrane, with a portion of MUC1 retained in cytosolic and perinuclear cell compartments. B. Image analysis for cell compartmentation using STAR morphology “Membrane Profile” calculation (see Star Methods). Image 1: cells with MUC1 preferentially localized at plasma membrane; Image 2: Harmony software identification of nucleus (blue) and plasma membrane (gold) in each cell; Image 3: STAR morphology “Membrane Profile” for the MUC1 predominant localization within membrane compartment C. STAR morphology “Membrane Profile” analysis of 203 compounds screened at 10 doses. R406 at most active concentrations reduced plasma membrane MUC1 abundance to a greater degree than most other compounds, as shown by deviation from the local regression. MUC1 IF signal intensity per cell (normalized to JQ1 minus DMSO-treated controls) plotted vs. DMSO-normalized MUC1 predominance in plasma membrane region as calculated using the STAR morphology “Membrane Profile” module. Horizontal and vertical dashed lines delineate mean DMSO values +/− 2*SD for both plotted parameters. Local regression was calculated by locally estimated scatterplot smoothing (loess) method +/− 95% confidence interval (gray-shaded).

Figure 4.. In vivo, R788 reduces excess MUC1 from lung epithelia of mice with ALI

A. Immunofluorescence images from lung tissue sections stained with MUC1 (green), Phalloidin (yellow), and DAPI (grey) demonstrate that ischemia-reperfusion (I/R)-induced remote ALI resulted in increased MUC1 in lung epithelium. Treatment with Fostamatinib over the course of 10 days suppressed MUC1 levels in mouse lung epithelium. B. Single cell tissue analysis, based on immunofluorescence of MUC1 and phalloidin (panel 1). In each image (panel 1) nuclei were identified based on DAPI staining (rainbow colors represent different cell nuclei in panel 2). The cell bodies were identified based on phalloidin staining surrounding each nucleus (orange cell borders, panel 3). Lastly, MUC1 IF intensity (green on panel 3) and Phalloidin intensities were calculated within each cell body. C. Bar graph ratio of MUC1/Phalloidin intensities in all cells of tissue sections from sham-treated mice and mice subjected to I/R-induced ALI, treated either with or without Fostamatinib. Average MUC1 intensity values per cell were normalized to the average phalloidin levels. Mean ± SD (n = 3 mice/condition/dose). ns, *p < 0.05 **p < 0.01 ***p < 0.001.